IL-6 promotes nonthyroidal illness syndrome by blocking thyroxine activation while promoting thyroid hormone inactivation in human cells

Abstract

Nonthyroidal illness syndrome (NTIS) is a state of low serum 3,5,3' triiodothyronine (T₃) that occurs in chronically ill patients; the degree of reduction in T₃ is associated with overall prognosis and survival. Iodthyronine deiodinases are enzymes that catalyze iodine removal from thyroid hormones; type I and II deiodinase (D1 and D2, respectively) convert the prohormone thyroxine T₄ to active T₃, whereas the type III enzyme (D3) inactivates T₄ and T₃. Increased production of cytokines, including IL-6, is a hallmark of the acute phase of NTIS, but the role of cytokines in altered thyroid hormone metabolism is poorly understood. Here, we measured the effect of IL-6 on both endogenous cofactor-mediated and dithiothreitol-stimulated (DTT-stimulated) cell sonicate deiodinase activities in human cell lines. Active T₃ generation by D1 and D2 in intact cells was suppressed by IL-6, despite an increase in sonicate deiodinases (and mRNAs). N-acetyl-cysteine (NAC), an antioxidant that restores intracellular glutathione (GSH) concentrations, prevented the IL-6-induced inhibitory effect on D1- and D2-mediated T₃ production, which suggests that IL-6 might function by depleting an intracellular thiol cofactor, perhaps GSH. In contrast, IL-6 stimulated endogenous D3-mediated inactivation of T₃. Taken together, these results identify a single pathway by which IL-6-induced oxidative stress can reduce D1- and D2-mediated T₄-to-T₃ conversion as well as increasing D3-mediated T₃ (and T₄) inactivation, thus mimicking events during illness.

Figures

Figure 1. Changes elicited by IL-6 in HEK-293 cells transiently expressing D2.

IL-6 concentrations, as observed in critically ill patients, inhibited D2 deiodination by intact cells (A) in a dose-dependent fashion (B), an effect already observed after 2 hours of incubation (C). The effects of IL-6 on intact cell T3 production were unaffected by low FT4 (D) or elevated rT3 levels (E). In contrast, similar doses of IL-6 enhanced D2 activity on DTT-stimulated assay in sonicated cells (F). Data are mean ± SD of at least 3 independent experiments. The net iodide release in this system is specific and equivalent to T3 production (5). *P < 0.001 versus control.

Figure 2. Changes elicited by IL-6 in cells expressing endogenous D2.

IL-6 decreased D2-catalyzed T4-to-T3 conversion in MSTO-211 cells (A), whereas it enhanced D2 maximal velocity in the DTT-stimulated assay in cell sonicates (B). The IL-6–induced increase in D2 protein was paralleled by an increase in its respective mRNA, which was blocked by preincubation with MAPK cascade inhibitors U0126 (ERK pathway) and SB203580 (p38 pathway) (C). DIO2 mRNA levels of control cells were set as 1. Data are mean ± SD of at least 3 independent experiments. *P < 0.001 versus control.

Figure 3. NAC abolishes the inhibitory effect of IL-6 on D2-mediated T3 production in MSTO-211 cells in a dose-dependent manner.

2 mM NAC abolished the effect of 500 ng/l IL-6 (A), but higher amounts of NAC (20 mM; B) were necessary to overcome the effect of 1,000 and 2,000 ng/l IL-6. IL-6 induced DIO2 mRNA, and this effect was not blocked by 2 mM NAC (C). DIO2 mRNA levels of control cells were set as 1. Data are mean ± SD of at least 3 independent experiments. *P < 0.0001 versus control.

Figure 4. IL-6 decreases D1-catalyzed T4-to-T3 conversion in HepG2 cells.

IL-6 inhibition of D1-catalyzed T3 production in intact cells was dose-dependent (A) and already observed after 2 hours of incubation (B). The effect of IL-6 was not altered by low FT4 levels (2 pM; C) and was abolished by the addition of 2 mM NAC to culture media (D). In contrast, IL-6 stimulated D1 activity in the DTT-stimulated assay in HepG2 cell sonicate (E). DIO1 mRNA levels in HepG2 cells were induced by IL-6, and this effect was blocked by U0126 or SB203580, but not by 2 mM NAC (F). DIO1 mRNA levels of control cells were set as 1. Data are mean ± SD of at least 3 independent experiments. *P < 0.001 versus control.

Figure 5. IL-6 induces D3 expression in MCF-7 cells.

(A) Chromatographic patterns of the products of D3-mediated T3 deiodination, determined by Sephadex LH-20 columns (top) or paper chromatography (bottom). IL-6 augmented intact cell T3 inactivation by D3 in a dose- and time-dependent fashion (B and C). D3 activity returned to control levels in the presence of NAC (D). D3 activity in cell sonicates and DIO3 mRNA levels were induced in the presence of 500 ng/l IL-6 (E and F). The effect of IL-6 on DIO3 mRNA levels was blocked by U0126 and SB203580, but not by 2 mM NAC. DIO3 mRNA levels of control cells were set as 1. Data are mean ± SD of at least 3 independent experiments. *P < 0.001 versus control.

Figure 6. IL-6 induces ROS generation in HEK-293, MSTO-211, HepG2, and MCF-7 cell lines in a dose-dependent fashion.

Cells were stained with 5 μM CM-H2-DCFDA and analyzed by fluorescence emission. Graphs express the normalized fluorescence intensity versus basal level. *P = 0.01 versus control.

Figure 7. H2O2 mimics the effects of IL-6 on D1, D2, and D3 deiodination in intact cells.

H2O2 decreased D1 (HepG2; A) and D2 (MSTO-211; D) and enhanced D3 (MCF-7; G) activities, as measured in intact cells, in a dose-dependent fashion; its effect was abolished in the presence of 2 mM NAC (B, E, and H). H2O2 induced DIO1, DIO2, and DIO3 mRNA levels, and this effect was blocked by U0126 and SB203580 and by 2 mM NAC (C, F, and I). DIO1, DIO2, and DIO3 mRNA levels of control cells were set as 1. Data are mean ± SD of at least 3 independent experiments. *P < 0.0001 versus control.

Figure 8. IL-6 reduces intracellular GSH levels in HEK-293, HepG2, MSTO-211, and MCF-7 cell lines.

(A) Effect of IL-6 on intracellular and extracellular GSH levels in various cell lines. (B) GSH (4 mM) prevented inhibition of intact cell D3 activity, but not that of D1 or D2, in HEK-293 cells transiently expressing these enzymes. (C) The effect of IL-6 in HEK-293 cells transiently expressing D3 was dose dependent and blocked in the presence of 4 mM GSH. Data are mean ± SD of at least 3 independent experiments. *P = 0.001 versus control.

Figure 9. Proposed mechanism for the effects of IL-6 resulting in NTIS.